Cells interact with structurally distinct types of extracellular matrix in different tissues at different stages of embryonic development and during adult wound repair. This project focuses on addressing the following major questions concerning the mechanisms of these cell-extracellular matrix interactions: 1. How do cells assemble a three-dimensional (3D) extracellular matrix, particularly one that is primarily based on fibronectin fibrils? 2. What are the differences in cell adhesive structures and biological responses between 2D and 3D matrices, as well as between different types of 3D matrices characteristic of different in vivo microenvironments? 3. What unique mechanisms control cell behavior in 3D microenvironments, especially those without oriented topological cues? Cells produce fibronectin-based fibrillar matrices using integrins and cellular contractility. We completed a study testing our hypothesis that point mutations in the cytoplasmic domain of the beta-1 integrin could identify specific sites with distinct functions in matrix assembly, signaling, cell adhesion, or cell spreading. We showed that certain integrin point mutants are markedly defective in their capacity to mediate assembly of a fibronectin matrix, while others alter cell spreading. We could rescue defective cell spreading by expressing constitutively active Akt, whereas matrix assembly is not rescued. Certain integrin point mutants also show defects in integrin activation and loss of the adapter/regulatory protein talin from integrin cell adhesion complexes. We could mimic the effects of these integrin mutations on fibronectin matrix assembly by gene silencing of talin 1 plus talin 2. We extended this work to identify a role for the talin rod domain in matrix assembly by mediating integrin connection to the cytoskeleton. We developed a dominant-negative inhibitor using the talin head domain to interfere with the ability of full length talin to mediate integrin connection to the actin cytoskeleton. This study established that specific cytoplasmic domain sites in a single integrin subunit can differentially modulate distinct integrin functions, and for the first time implicated talin in the process of fibronectin matrix remodeling. These studies are being extended to study cytoplasmic domain interactions of tumor cells in collaboration with Dr. Allison Berrier at LSU. We previously published evidence for the importance of the three-dimensionality of the extracellular matrix surrounding fibroblasts in a variety of cell biological functions including signal transduction, migration, and proliferation. We initiated tests of two hypotheses concerning the mechanisms of cell migration in 3D settings. Cells are known to be surrounded by biochemically and structurally distinct matrices in vivo, e.g., a matrix rich in fibronectin fibrils during early craniofacial neural crest migration versus a collagen-rich matrix with varying crosslinking in adult connective tissue. We hypothesize that (1) three-dimensionality and differences in biochemical composition or crosslinking can combine to determine altered requirements for cytoskeletal and proteolytic responses needed for cell migration within different 3D matrices. We further hypothesize that (2) the role of myosin IIA (and possibly IIB) may differ in 3D versus 2D settings, as well as in different types of 3D environments. Our preliminary data suggest that depending on the type of 3D matrix (cell-derived versus collagen gels), myosin II isoforms may or may not be required for effective cell migration;this dichotomy in 3D contrasts with its migratory down-regulatory function in 2D cell culture, where ablating it stimulates migration. We are testing for morphological differences between cell adhesions in various 3D environments, comparing 3D matrices comprised of collagen, fibrin, or cell-derived matrix components. Our previous studies implicated the cytoskeletal protein tensin in fibronectin matrix assembly. In collaboration with Katherine Clark and David Critchley, we have continued to compare the functions of the three large tensin isoforms. We find evidence for both overlapping and distinct localization and function, with Tensin 2 enrichment in dynamic focal adhesions at the leading edge of the cell. We are also testing roles of this tensin in 3D collagen gel remodeling. We are continuing to develop methods to visualize cell-surface and cytoskeletal molecular complexes and dynamics in 3D matrices. This technically challenging microscopy technology will be important to develop further in order to allow direct comparisons of cellular functions in 2D versus 3D environments. Understanding whether the extent and nature of requirements for a specific protein differ in 2D and 3D appears important, since initial conclusions from in 2D vitro studies may differ under 3D or in vivo conditions.